The immune system plays an important role in defense against specific microorganisms, for example viruses, specific fungi and bacteria, as well as in recognizing and inhibiting and/or eradicating tumor cells. Vaccinations are a long-known method of activating the immune system. Vaccination and immunization is the introduction of a non-virulent agent into a subject, in which the agent elicits the subject's immune system to mount an immunological response. Often, vaccine antigens are killed or attenuated forms of the microbes which cause the disease. The presence of non-essential components and antigens in these killed or attenuated vaccines has encouraged considerable efforts to refine vaccine components including developing well-defined synthetic antigens using chemical and recombinant techniques. The refinement and simplification of vaccines, however, has led to a concomitant loss in potency. Low-molecular weight synthetic antigens, though devoid of potentially harmful contaminants, are often not sufficiently immunogenic by themselves and do not produce an adequate immune response.
The immunogenicity of an antigen can be increased by administering it in a mixture with substances called adjuvants. Adjuvants increase the response against the antigen either by directly acting on the immunological system or by modifying the pharmacokinetic characteristics of the antigen, resulting in an increased interaction time between the antigen and the immune system. Additionally, the addition of an adjuvant can permit the use of a smaller dose of antigen to stimulate a similar immune response, thereby reducing the production cost of a vaccine.
Currently the most widely adjuvants used in humans are Aluminum salts. Aluminum salts have been useful for some vaccines like hepatitis B, diphtheria, tetanus, and toxoid; however, they are not useful for others like rabies, MMR, and typhoid. In addition, Aluminum salts fail to induce cell-mediated immunity, result in the induction of granulomas at the injection site and vary in effectiveness between batches of alum preparations.
Detection of specific viral pathogen-associated molecular patterns (PAMPs) by host pathogen recognition receptors (PRRs) is the first event in the defense against virus infection (1-3). During infection with RNA viruses, the RIG-I-like receptors (RLRs) RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated protein 5) bind viral PAMPs present in the infected cell and initiate a signaling cascade mediated by the mitochondrial antiviral signaling molecule (MAVS), which culminates in the production of type I IFNs and other antiviral genes (4-6). This primary host response to infection is essential to control virus replication and to initiate protective antiviral immunity (7). RNA motifs that effectively trigger this pathway are disclosed herein, which can, in certain embodiments, be used to generate synthetic RLR ligands for strong induction of antiviral responses in the context of vaccination or antiviral therapies.
5′-di- or -triphosphates associated with double strand RNA (dsRNA) or with single strand RNA, with or without poly U/UC ssRNA stretches, trigger RIG-I stimulation (1, 3, 8-10). These structures are present in influenza A virus (IAV) (8, 11), Sendai virus (SeV) (12), Hepatitis C virus (HCV) (13-15), and reovirus (16, 17). MDA5 ligands are much less characterized and are presumed to be complex secondary RNA structures (18, 19).
During infection with viruses that are adapted to the host, viral-encoded proteins interfere with RLR activity allowing the virus to reach high titers prior to the onset of the antiviral response (20-22). The delay in detection of viral structures that are normally present in the viral genomes during natural infections indicates that additional factors are required for the effective triggering of antiviral responses in vivo. During infection with SeV, potent stimuli for the initiation of the antiviral response is provided by immunostimulatory defective viral genomes (iDVGs) generated during virus replication at high titers (23-25). SeV iDVGs trigger RLR signaling and initiate strong antiviral immunity both in vitro and during natural infections in vivo (26, 27). SeV iDVGs belong to the copy-back type of RNA DVGs and are produced when the viral polymerase is released from the template strand during replication and copies back the nascent strand (28). iDVGs are unable to replicate in the absence of helper virus as they lack essential replication machinery, and are not transcribed into proteins as they are flanked by the antigenomic promoter (29-32). A copy-back iDVG of 546 nucleotides (DVG-546) predominant in laboratory stocks of SeV strain Cantell (SeV C) has been characterized to be a strong trigger of RLRs signaling (12, 23, 26, 33). Although this activity largely depends on 5′triphosphates and the presence of dsRNA structures, prior to the instant disclosure, it was unclear whether additional RNA motifs optimize the immunostimulatory potential DVG-546 contributing to its efficient recognition even in the presence of virus-encoded antagonists of the antiviral response (25, 34).